PROJECT SUMMARY Swine are important in biomedical research for the study of human diseases that are poorly recapitulated by rodent species, for the development and testing of preclinical therapeutics in humanized disease models and as potential sources of xenogeneic or allogeneic organs and tissues. However, the creation and propagation of biomedical swine is plagued by inefficiencies related to animal development, reproduction and lethal phenotypes. Production breeding programs for swine model propagation are inadequate when the models have severe disease-associated phenotypes that reduce long-term viability, the ability to sexually reproduce or segregate numerous alleles. For regenerative medicine purposes, the development and propagation of organogenesis-deficient animals also requires an alternative to standard breeding. Our solution is to develop a platform technology based on germline stem cell transplantation (GST) and blastocyst complementation in swine to rescue the germline of valuable lines and permit for the first time efficient propagation of congenital disease and organogenesis-deficient alleles. Application of GST or blastocyst complementation relies on the generation of a strain of pigs that cannot produce their own gametes. In Phase I, we generated germ cell-deficient pigs by creating homozygous null mutations in the Deleted-in-Azoospermia-like (DAZL) gene. We've also established a breeding herd of heterozygous DAZL animals that are phenotypically normal and fertile. In this Phase II grant, we will optimize the conditions for GST in DAZL-null boars by evaluating germ cell engraftment and sperm characteristics after transplantation of limiting dosages of germline stem cells. Secondly, since germline stem cells from severe disease models could be limited due to high morbidity, we will quantify germline stem cell yield from 1wk to 10 week-old testes and develop strategies for expansion of these cells. As proof of concept, we will conduct germline transplantation from our severe model of dilated cardiomyopathy (DCM) that is inefficiently produced by standard breeding. By rescuing the germline of homozygous DCM boars, we will double our production of this model without the need to expand our sow herd. In the last aim of this grant, we will use blastocyst complementation to demonstrate phenotypic rescue and fertility of our RAG2/IL2Rg knockout (SCID), immunodeficient swine. DAZL-null donor cells will rescue the immunodeficient phenotype but are unable to contribute to the germline of chimeras. Successful implementation of this approach would increase the production rate of SCID animals from the current 6.3% by intercross of heterozygotes to 100% with DAZL- enabled intercross of homozygotes. Hence, this proposal will have immediate impact on our ability to produce and sell two swine models with increased margins, DCM and SCID, while creating a novel DAZL breeding platform that numerous other models and organogenesis-deficient lines will rely on for propagation and scale- up. 0